627 629 blk

Fig. 1. RNA dot blots used to eliminate cell lines that produce intron-containing fusion transcripts. Duplicate RNA dot blots containing 10 |g total RNA were hybridized with lacZ and intron probes of the gene trap vector. 433 is a typical example of an intron-containing cell line.

3.3.3. RACE Cloning

The following protocol has been streamlined and contains a number of modifications to the original method described by Frohman, et al. (ref. 14). The most important of these are:

1. Alkaline hydrolysis of the RNA (required for efficient T-tailing with terminal deoxytransferase);

2. Synthesis of second-strand cDNA with Klenow instead of Taq polymerase; and

3. The use of microdialysis filters, which serves a dual role of removing primers/ buffers between steps and size-selecting informative cDNA fragments above 300 bp in length.

By way of example, Table 1 lists the oligos used for RACE cloning from cell lines obtained with the pGT1.8Pgeo and pGT1.8TM vectors.

1. Spin down 5-10 |g of total RNA in an Eppendorf tube, wash RNA pellet twice with 70% ethanol, and partially dry pellet in Speed Vac. Repeat this step, since it is important to remove any residual urea. Resuspend RNA sample on ice in 10 |L of DEPC-treated water. Add 1 |L of 10 ng/|L primer 1, and heat for 5 min at 70°C. Cool on ice and spin briefly.

2. Set up first-strand reaction by adding:

1 |L of Superscript II RT.

Incubate first-strand reaction at 37°C for 1 h.

3. To improve the efficiency of the tailing reaction, hydrolyze RNA by adding 2.2 |L of 1 M sodium hydroxide for 20 min at 65°C. Neutralize with 2.2 |L of 1 M hydrochloric acid.

Table 1

Primer Combinations Used in 5' RACEa

Primer Gene trap vector, pGT1.8geo Secretory trap vector, pGT1.8tm






aXbaI and KpnI sites used in cloning the RACE products are underlined.

4. Microdialyze sample on a 0.025-|im filter floating in a Petri dish of TE for 4 h. Transfer the remainder of the sample to an Eppendorf tube, and wash the filter with water to bring the final volume to 20 ||L.

5. Set up tailing reaction by adding 6 ||L of TdT buffer (5X) and 2 |L of 2 mM dATP. Incubate for 2 min at 37°C. Add 2 |L of TdT enzyme, and incubate for a further 5 min. Stop the reaction by heating to 70°C for 2 min.

6. Carry out second-strand synthesis by adding the following to 15 |L of tailed cDNA:

2 |L restriction buffer M (10X). 1 |L dNTPs (10 mM). 1 |L primer 2 (10 ng/mL). 1 |L Klenow enzyme. Incubate for 30 min at room temperature, 30 min at 37°C, and 5 min at 70°C.

7. Microdialyze for 4 h on a 0.1-|im filter. Recover cDNA from filter into a final volume of 37 ||L of H2O.

8. For the first-round PCR reaction, add the following to 37 |L of sample:

5 |L 10X AmpliTaq buffer

1 |L primer 3 (100 ng) 1 |L primer 4 (100 ng) 1 |L AmpliTaq Carry out 30 cycles using the following parameters: Denature 94°C for 1.5 min

Anneal 60°C for 1.5 min

Extend 72°C for 3.0 min

9. Microdialyze for 4 h on a 0.1-|im filter to remove smaller, uninformative PCR products and excess primers. Recover sample from filter.

10. Perform second-round PCR using 5 |L of the first-round PCR reaction and:

5 |L PCR buffer

1 pL primer 3 (100 ng/mL) 1 pL primer 5 (100 ng/mL) 1 pL AmpliTaq 37 pL H2O

Use same cycle parameters as first-round PCR, but use a hot start. After the final cycle, polish the second-round PCR products by adding 50 ng of each primer, 0.5 pL of dNTPs, and 0.5 pL of Amplitaq. Perform one cycle using the following conditions: Denature 94°C for 1.5 min

Anneal 60°C for 1.5 min

Extend 72°C for 20.0 min

11. Microdialyze for 4 h on a 0.1-pm filter, and recover sample from filter. Analyze 5 pL of sample by gel electrophoresis and Southern blot hybridization (see Note 5). Digest the remainder of the sample with XbaI and KpnI.

12. Following digestion, extract the sample twice with phenol/chloroform, extract once with chloroform, and precipitate the RACE products on ice for 10 min by adding 5 pg of glycogen, 25 pL of 10 M ammonium acetate, and 300 pL of etha-nol. Microfuge for 10 min, wash pellet with 70% ethanol, partially dry pellet in Speed Vac and resuspend in 20 pL of TE.

13. Ligate 100-200 ng of XbaI/KpnI-digested RACE products with 50-100 ng XbaI/ KpnI-digested plasmid DNA in a final volume of 15 mL at room temperature for at least 2 h:

4-8 pL Digested PCR products (100-200 ng) 1 pL Digested plasmid DNA (100 ng) 1.5 pL Ligation buffer (10X) 1 pL T4 DNA ligase 3.5-7.5 pL H2O

14. Transform into competent bacteria, and screen colonies for desired inserts (see Note 6).

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